Efficient gene transduction in cardiomyocytes is a task that can be accomplished only by viral vectors. Up to now, the most commonly used vectors for this purpose have been adenoviral-derived ones. Recently, it has been demonstrated that lentiviral vectors can transduce growth-arrested cells, such as hematopoietic stem cells. Moreover, a modified form of lentiviral vector (the ‘advanced’generation), containing an mRNA-stabilizer sequence and a nuclear import sequence, has been shown to significantly improve gene transduction in growth-arrested cells as compared to the third-generation vector. Therefore, we tested whether the ‘advanced’generation lentivirus is capable of infecting and transducing cardiomyocytes both in vitro and in vivo, comparing efficacy in vitro against the third-generation of the same vector. Here we report that ‘advanced’generation lentiviral vectors infected most (> 80%) cardiomyocytes in culture, as demonstrated by immunofluorescence and FACS analyses: in contrast the percentage of cardiomyocytes infected by third-generation lentivirus was three-to four-fold lower. Moreover,‘advanced’generation lentivirus was also capable of infecting and inducing stable gene expression in adult myocardium in vivo. Thus,‘advanced’generation lentiviral vectors can be used for both in vitro and in vivo gene expression studies in the cardiomyocyte.